Abstract

Fibrin, a product of the blood coagulation cascade, accompanies many type 1 immune responses, including delayed-type hypersensitivity, autoimmunity, and graft rejection. In those settings, fibrin is thought to exacerbate inflammation and disease. Here, we evaluate roles for coagulation during infection with Toxoplasma gondii, a pathogen whose control requires robust type 1 immunity. We establish that fibrin prevents infection-stimulated blood loss, thereby performing a protective function that is essential for survival. Remarkably, fibrin does not simply protect against vascular damage caused directly by the infectious agent, but rather, protects against hemorrhage evoked by interferon-γ, a critical mediator of type 1 immunity. This finding, to our knowledge, is the first to document a beneficial role for coagulation during type 1 immunity, and suggests that fibrin deposition protects host tissue from collateral damage caused by the immune system as it combats infection.

Introduction

During the coagulation cascade, fibrin is generated by the action of thrombin on fibrinogen. Although best appreciated for its roles in vascular hemostasis, coagulation leading to fibrin deposition also frequently accompanies type 1 immune responses, including delayed-type hypersensitivity (1–3), autoimmunity (4–10), and graft rejection (11–13). Prior studies indicate that fibrin, and other coagulant products, can exacerbate inflammation and disease in those settings (1–16). Coagulation also frequently accompanies infectious disease, and apparently can exacerbate pathology in that context as well (16). Indeed, administration of activated protein C, a natural anticoagulant protein, reduces septic mortality in humans (17). While these and other studies have documented pathological roles for coagulation during immunity and infection, beneficial functions for immune-associated coagulation have yet to be established.

Toxoplasma gondii is a widely distributed, obligate intracellular, protozoan parasite (18, 19). Natural infection is acquired by ingestion of encysted organisms, which are released in the digestive tract, invade intestinal tissues, and then disseminate. In mouse models, T. gondii infection elicits a robust type 1 immune response, which eradicates most parasites within 2 wk of infection. Nevertheless, some organisms escape immune surveillance, encyst, and enter a dormant state from which they can emerge if immunity wanes, as frequently occurs in humans suffering from the acquired immunodeficiency syndrome.

Here, we evaluate roles for fibrin in the context of the strong type 1 immune response prompted by T. gondii infection. In contrast to prior studies of immune-associated fibrin deposition, we demonstrate that coagulation performs a critical protective function in the setting of T. gondii infection by suppressing hemorrhage evoked by IFN-γ, a critical mediator of type 1 immunity. Thus, while coagulant products have the potential to function pathologically, our data indicate that immune-associated coagulative responses can also protect host tissue from collateral damage caused by the immune system as it combats infection.

Materials And Methods

Mice.

6–10-wk-old male mice were used for all experiments. C57BL/6 and IFN-γ–deficient mice were purchased from Taconic and The Jackson Laboratory, respectively. Fibrinogen-deficient mice (reference 20; backcrossed seven generations to C57BL/6 mice) were generously supplied by Jay Degen (Children's Hospital Medical Center, Cincinnati, OH) and were bred at the Trudeau Institute Animal Breeding Facility. Animals were housed in a specific pathogen-free facility and cared for according to the Trudeau Institute Animal Care and Use Committee guidelines.

To evaluate the function of this infection-stimulated coagulative response, we treated mice with warfarin, an orally available pharmaceutical used clinically for the long-term anticoagulation of thrombosis-prone humans. Supplementing drinking water with 2 mg/L warfarin did not impair the growth or survival of uninfected mice, but was clinically effective, as it suppressed T. gondii–stimulated fibrin deposition to below the detection limit of our assay (unpublished data). Strikingly, warfarin-treated mice acutely succumbed to T. gondii infection, dying around the time of the peak coagulative response of untreated animals (Fig. 2
A). That finding suggested that coagulation might function protectively during T. gondii infection.

Fibrin may protect against infection-triggered mortality via a number of plausible mechanisms. As levels of fibrin and parasitemia correlated (Fig. 1), we considered the possibility that coagulation might function to physically limit parasite dissemination (14). However, parasite numbers within liver, intestine, Peyer's patch, spleen, and brain were not significantly increased in fibrinogen-deficient mice (Fig. 3
, and unpublished data).

Mice lacking fibrin exhibit neither increased parasite burdens nor suppressed anti-parasite immunity. Fibrinogen-deficient (white bars) and littermate control fibrinogen-heterozygous mice (black bars) were infected with T. gondii. Parasite numbers (p30 mRNA in liver), IFN-γ (protein in sera, mRNA in liver), nitric oxide (nitrite/nitrate in sera), inducible nitric oxide synthase (iNOS, mRNA in liver), IL-10 (mRNA in liver), and TNF-α (mRNA in liver) were assayed on days 4 and 8 after infection. There were no significant differences in the levels of any of the measured parameters. The data shown was compiled from four independent experiments. Bars depict average ± SD of 8 mice per group (Day 0), 4 mice per group (Day 4), or 14 to 16 mice per group (Day 8).

In experimental models, acute mortality during T. gondii infection can result from either a failure to control parasite numbers, or excessive immunopathology (19). During the course of our studies we noted exacerbated gross pathology in liver tissue of T. gondii–infected fibrinogen-deficient mice (Fig. 4
B). Microscopic examination revealed intense infiltration of mixed inflammatory cells and areas of hepatocellular necrosis in livers of T. gondii–infected control mice (Fig. 4 C). In fibrinogen-deficient mice, infiltrates tended to be more diffuse, and necrosis was more extensive and frequently associated with hemorrhage (Fig. 4 D).

Fibrin protects against infection-stimulated, IFN-γ–mediated hemorrhagic immunopathology. (A) Wild-type C57BL/6 mice were infected with T. gondii. On the indicated days, blood was collected and hematocrits, red cell numbers (unpublished data), and hemoglobin levels (unpublished data) were determined using a Coulter Counter (Beckman Coulter). Compared with uninfected mice, each of those parameters were significantly reduced by day 10 after infection (P < 0.001; n = 5 mice per group). (B) Fibrinogen-deficient and littermate control heterozygous mice were infected with T. gondii or were sham infected. On day 8 after infection, hematocrits were significantly reduced in T. gondii–infected control mice (*P < 0.02; n = 5 mice per group). Compared with infected control mice, T. gondii–infected fibrinogen-deficient mice displayed even greater hematocrit reductions (**P < 0.005; n = 5 mice per group). (C) Wild-type C57BL/6 and C57BL/6-backcrossed IFN-γ–deficient mice were infected with T. gondii. 8 d later, hematocrits were significantly reduced in T. gondii–infected wild-type control mice (*P < 0.005; n = 5 mice per group), but not in the IFN-γ–deficient mice. (D) Wild-type C57BL/6 received a single injection (1 mg, intraperitoneal) of control rat IgG1 mAb or anti–IFN-γ mAb (αIFNγ). The next day, mice were sham infected or infected with T. gondii. 8 d later, hematocrits were significantly reduced in T. gondii–infected control mAb-treated mice (*P < 0.008; n = 5 mice per group), but not in the anti-IFN-γ–treated mice. (E) Fibrinogen-deficient and littermate control mice received control rat IgG1 mAb or anti-IFN-γ mAb (1 mg, intraperitoneal), and were infected with T. gondii the following day. Hematocrits were measured at day 8 after infection. In both sets of mice, anti-IFN-γ mAb treatment significantly protected against hematocrit reductions (*P < 0.01; n = 4 mice per group). Notably, the hematocrits of anti-IFN-γ mAb-treated fibrinogen-deficient and littermate control mice were not significantly different. Similar results to those shown were replicated in two (A) or more (B–E) independent experiments.

We next assessed how infection evokes a coagulative response. T. gondii infects all nucleated cells (18), and thus has the potential to directly damage vascular cells, thereby causing hemorrhage and activating a coagulative response. However, parasite-stimulated immunopathology could also prompt hemorrhage leading to coagulation, as levels of IFN-γ and its potentially destructive downstream product, nitric oxide, both peak around the time of the acute death of coagulation-impaired T. gondii–infected animals (Fig. 1, and unpublished data). Notably, IFN-γ also up-regulates cellular expression of procoagulant molecules (26–28), thereby potentially amplifying fibrin production.

In contrast to those prior reports, our studies demonstrate that coagulation can also function protectively during immunity. Specifically, we found that fibrin can prevent infection-stimulated blood loss, thereby performing a protective function that is essential for survival. Remarkably, fibrin does not simply protect against vascular damage caused directly by the infectious agent, but rather, protects against hemorrhage evoked by IFN-γ, a critical mediator of type 1 immunity. Notably, IFN-γ also stimulates expression of genes that favor and/or stabilize fibrin deposition (26–28), suggesting that type 1 immunity may have evolved means to locally up-regulate coagulant activity, thereby preemptively protecting against its own destructive power.

Our observations indicate that the pathological potential of immune-associated coagulation is counter-balanced by a critical protective function. Such findings help to explain why type 1 immunity frequently elicits fibrin deposition (1–13), and suggest that clinicians should be cautious in ascribing pathological assessments to immune-associated fibrin deposits. While the inflammatory activities of fibrin and other coagulant products may function pathologically in certain contexts (1–16), immune-associated coagulative responses can also function beneficially, by protecting against immunopathology.

Notably, our data do not exclude the possibility that fibrin may also function protectively through direct effects on immunity and/or inflammation. In comparison to littermate control mice, there were no significant differences in pathogen dissemination or levels of immune mediators in T. gondii–infected fibrinogen-deficient mice (Fig. 3). However, the robust type 1 immune response that accompanies T. gondii infection may have masked immune-related impacts of fibrin-deficiency. Indeed, while own prior work demonstrated that thrombin can stimulate fibrin-dependent secretion of inflammatory cytokines/chemokines in vivo (30), subsequent studies found that endotoxin- or T cell–stimulated secretion of those same cytokines/chemokines is fibrin-independent (unpublished data). Thus, we suspect that infection-stimulated immunity may well mask fibrin's inflammatory activities during T. gondii infection.

Our data suggest that fibrin-unrelated coagulation deficiencies, whether genetic or pharmacologic, may also exacerbate immunopathology. As such, we are now exploring the generality of our findings. We are also working to define precisely how IFN-γ evokes pathology necessitating a protective coagulative response. We anticipate that those studies may suggest novel therapies for pathologies characterized by extensive fibrin deposition, including transplantation and many forms of autoimmunity, and may also impact treatments for the numerous virulent human pathogens that elicit deleterious coagulative and/or hemorrhagic responses.

Acknowledgments

We are grateful to Drs. Robert North, Troy Randall, Wayne Hancock, and Terri Laufer for critical reading of this manuscript. We also wish to thank Paula Lanthier and Jean Brennan for technical assistance, and Pamela Scott Adams for help with real-time PCR. We are indebted to the employees of the Trudeau Institute Animal Breeding and Maintenance Facilities for dedicated care of the mice used for these studies.

This work was supported by PHS Grant AI46571 (L.L. Johnson) and funds from Trudeau Institute.